Activity of the IL-4 promoter was shown to be regulated by multiple cis-acting elements. In this study, two additional regulatory elements, a CCAAT element and a 15-nucleotide element homologous to the IFN- and virus-stimulation response element (ISRE), were identified in the human promoter region at -195 to -172. The ISRE-homologous sequence was shown to interact with two nuclear proteins, IRF-2, a transcriptional repressor of the IFN genes, and an NF-1-like factor. Mutations of the ISRE site increased overall IL-4 promoter activity twofold, suggesting a possible negative role of IRF-2 in the regulation of IL-4 transcription. The CCAAT element was found to interact with NF-Y, a nuclear factor essential for expression of MHC class II genes. Mutations of the CCAAT element at -195 to -172 resulted in a substantial decrease of IL-4 promoter activity. Furthermore, NF-Y was also found to bind to the previously described NF-ATp binding site of the IL-4 promoter (-79 to -62, originally described as "P element"), and the previously described P-binding complex NF-P was shown to contain NF-Y. These findings suggest that NF-Y may play an important role in the regulation of IL-4 gene expression.